人真皮多能干细胞移植对脑缺血损伤修复的探讨
摘要
研究背景和目的:
中枢神经系统(central nervous system,CNS)损伤修复一直是困扰神经科学工作者的重大难题。传统观念认为中枢神经系统一旦损伤便不能恢复,但随着近年来神经科学的发展,尤其是干细胞相关研究的不断深入,人们逐渐认识到成年CNS再生是由神经元的固有特性(intrinsic properties)和其所处的环境(environmental cues)共同决定的。因此增加受损神经元的再生潜能并改善轴突再生的微环境便成为CNS损伤修复研究的新的楔入点。
神经损伤后修复的难点之一是神经元再生困难,随着近年来干细胞研究的迅猛发展,组织和细胞移植治疗成为学者们最为关注的方法之一,而选择合适的种子细胞则是细胞移植治疗的核心问题。目前用于移植的种子细胞供体主要有胚胎干细胞、神经干细胞(neural stem cells,NSC),以及其他具有成神经分化潜能的成体干细胞。尽管这些细胞在CNS损伤修复移植治疗中均显示出不同程度的积极作用,但由于存在细胞来源局限、取材不易、免疫排斥以及伦理道德等诸多方面的问题,临床应用受到很大限制。因此,寻找新的种子细胞来源成为当务之急。皮肤是人体最大的器官,包括来源于三个胚层的多种不同的组织细胞,自我更新和再生修复速度快,是成体干细胞分离和移植研究的良好对象。真皮多能干细胞(skin-derived precursors,SKPs)是一种新近分离成功的具有高度增殖能力和多向分化潜能的间充质干细胞,具有分化为脂肪细胞、肌细胞及神经细胞的潜能。相对于其他移植供体细胞而言,SKPs具有来源丰富、容易获得、可自体移植和具有更少的“谱系倾向性”等优点,又避免了胚胎伦理学、来源不足和异体排斥等弊端。相关研究表明,SKPs移植治疗脊髓损伤时,不仅能外在补充缺失神经元,同时可通过分化为Schwann细胞促进轴突再生及髓鞘化,显示了SKPs在CNS损伤修复中的良好应用前景。
研究内容和方法:
为了解SKPs移植对脑缺血大鼠中枢神经损伤的修复功能及神经保护作用,本实验进行了下列相关研究。
1.利用悬浮成球及单层贴壁相结合的细胞培养方法,分离成人包皮组织,获得真皮来源细胞,无血清条件下添加B27及生长因子bFGF和EGF使其悬浮生长,培养数日后去除B27及生长因子,添加血清使细胞贴壁生长并快速扩增。获得SKPs细胞后,应用免疫化学方法检测其表面标志物CD29、CD34和Nestin的表达。
2.线栓法制作大鼠大脑中动脉缺血再灌注损伤模型,CM-DiI标记SKPs后,通过股静脉移植到大鼠体内。通过抓握实验、平衡木行走实验及水迷宫实验对实验大鼠进行移植前后神经功能评分。
3.免疫荧光法检测各组实验大鼠神经细胞(NSE、GFAP)相关抗原的表达,观察移植SKPs在实验大鼠体内存活及向神经细胞分化情况。
4.利用TUNEL法分别检测各个时间点缺血对照组及移植组脑内神经细胞凋亡情况,镜下观察脑组织健患侧神经细胞凋亡数目。
结果:
1.采用悬浮成球方法分离培养人真皮来源SKPs,此时细胞扩增较慢,细胞贴壁生长后,细胞扩增速度加快,2-3d传代一次。根据SKPs特异性标志,对其进行免疫染色鉴定,体外单层贴壁培养的真皮多能干细胞,CD29和Nestin呈阳性表达,CD34阴性表达。
2.通过抓握实验、平衡木行走实验及水迷宫实验在SKPs移植后1、3、5、7、14天五个时间点对正常组、脑缺血对照组及SKPs移植组大鼠进行神经功能评分。与正常组比较,缺血后1-14天,模型组各项神经功能检测指标均明显降低(P<0.05);观察期内有自发恢复特点,但仍低于正常组水平:与对照组比较,缺血后3、5、7、14d,SKPs移植组能够增加大鼠的抓握力(P<0.05);促进脑缺血大鼠平衡木行走的能力(P<0.05);与对照组比较缺血后5、7、14d能够明显促进脑缺血大鼠记忆功能的恢复(P<0.05)。
3. SKPs移植1天可见移植的SKPs主要分布在脑组织缺血侧,表明SKPs经股静脉移植后可在短时间内迁移到脑缺血区域,由于我们在免疫组化中应用的是小鼠抗人的GFAP单克隆抗体和兔抗人NSE单克隆抗体,这些表达GFAP和NSE的细胞同时表达CM-DiⅠ,因此GFAP和NSE阳性细胞来源于移植的SKPs,表明SKPs经移植进入实验大鼠体内仍保留向神经细胞分化的特性。为进一步增加脑组织中外源性神经干细胞数量提供了一条思路。
4.正常组和缺血对照组、SKPs移植组的健侧脑组织TUNEL染色只有零星的凋亡细胞。缺血对照组整个缺血区内均可见到凋亡细胞,大量成簇出现的凋亡细胞主要分布在梗死灶边缘区(皮层)的内侧。SKPs移植组凋亡细胞分布与缺血对照组相同,但皮层缺血半暗带区凋亡细胞数(57±15)与缺血对照组(102±7)比较明显减少(P<0.05)。
结论:
我们利用悬浮成球及单层贴壁相结合的培养方法成功地从人包皮组织中分离并扩增了SKPs。移植SKPs到脑缺血损伤大鼠体内能够存活并定向迁移至脑缺血部位并向神经细胞分化,同时可减少缺血区的神经细胞凋亡,促进大鼠脑缺血损伤后的神经功能恢复。结果提示移植的SKPs可能是通过向神经细胞的分化、抑制缺血区神经细胞的凋亡来促进缺血损伤后的神经再生和功能重建,SKPs有望成为干细胞移植治疗的新的种子细胞来源。
The recovery of central nervous system(CNS)after injury is all along a difficult problem that puzzles the neuroscientists.In the past,it was widely accepted that once CNS was injured,nothing could be done.Since recent two decades,numerous studies have been carried out by neuroscientists and it is concluded that CNS after injury can be recovered.The course of recovery based on two factors:intrinsic properties and environmental cues.So,it is aimed to promote the regeneration of neurons and provide a possible agreeable microenvironment for regeneration and recovery of CNS injury.
In regenerative medicine,some encourage results have been obtained.It is well known that cell replacement therapies show particular promise in the nervous system,and transplanted embryonic stem cells(ESCs)or neural stem cells (NSCs) have been shown to promote functional recovery in animal models,for example,spinal cord injury (SCI).Many stem cells (such as ESCs and NSCs) can be used to treat a lot of neurological diseases. Although the therapeutic potential of such transplants is clear,a number of problems, such as insufficient cell number, ethical issues, immunosup- pression, and so on,limit their application in clinic.In this background,a new type of stem cells called skin-derived precursors (SKPs) was discovered in adult mammalian skin.They can differentiate into cells of both neuroectodermal and mesodermal lineage,including(perhaps not limited to) neurons,glia,osteogenic cells and adipocytes.Compared with the above cells, SKPs, which own multi-oriented differentiative potentialand self-renewal capability,have an accessible,potentially autologous tissue source and can expand in vitro cell culture.So they have important therapeutic implications.
In order to determin if the SKPs have the effects of promoting the regeneration and function recovery of Ischemic brain injury. a series of studies were carried out as follows: (1) Combination of suspension and adherent culture method , digested adult dermal tissue, Serum-free conditions, Add B27 and growth factors bFGF and EGF to suspended growth. Immunochemistry to detect the surface markers CD29, CD34 and Nestin. (2) Middle cerebral artery occlusion (MCAO) models were produced by obstructing MCA using a single strand nylon thread, CM-DiI labeled cells, Through the femoral vein transplanted into rats. Neurological function was evaluated with beam-walking,bar-grasping and water maze. (3) Immunofluorescence assay (NSE, GFAP)-associated antigen expression of rat nerve cells. To investigate the survival and differentiation of skin-derived precursors transplanted into rat brain.(4) TUNEL kits were used to detect the control group and transplant brain tissue neural cell apoptosis. Microscopic observation of brain tissue in the number of neuronal apoptosis.
Main results are as following:
1. Combination of suspension and adherent culture , According to SKPs specific signs, Most cells expressed CD29 and Nestin instead of CD34b y immunofluorescent staining.
2. ischemia group rats and SKPs transplantation group rats showed severe neurological disfuction compared with sham-operated group(p<0.05). Rats receiving SKPs had a more rapid recovery of beam-walking,bar-grasping and water maze performance than ischemia group rats(p<0.05).
3. Twenty-four hours later, the transplanted SKPs can be detected mainly in the ischemic zone. These suggested that the transplanted SKPs can migrate to the ischemia zone through the femoral vein graft in a short time. The immunofluorescent staining results confirmed a part of NSE and GFAP positive cells expressed CM-DiI also. These data suggested that the number of nerve cells differentiated from SKPs. In order to further increase the number of exogenous neural stem cells provides a train of thought.
4. TUNEL: After 2 hours of MCA occlusion and 24 hours reperfusion, apoptotic cells exhibiting DNA fragmentation increased in the regions of caudoputaminal and cortex, groups of apoptotic cells were primarily localized to the inner boundary zone of the infarct. The number of apoptotic cells of SKPs transplantation group(57±15)decreased significantly compared with ischemia group(102±7)in the penumbra of cortex with light microscopic analysis (x400 field),p<0.05.
Conclusion: Combination of suspension and adherent culture , Skin-derived precursors (SKPs) were isolated from the adult dermal tissue,cultured and identified by their multilineage differentiation capacity. SKPs can survive in the brain of MCAO rats and were mainly distributed in the region around the ischemic focus and remained the differentiation characters of neuron cells. The results demonstrate that SKPs can improve the neurological function follwing focal cerebral ischemia in rats. Manifested as behavioral score was significantly higher、to reduce the number of neuronal apoptosis in Damage zone. The results suggest that transplant SKPs may be through to Differentiat into neural cells and inhibit neuronal apoptosis in ischemic area to promote nerve regeneration and functional reconstruction after ischemic injury,For clinical treatment of ischemic brain injury provides a new way.
中枢神经系统(central nervous system,CNS)损伤修复一直是困扰神经科学工作者的重大难题。传统观念认为中枢神经系统一旦损伤便不能恢复,但随着近年来神经科学的发展,尤其是干细胞相关研究的不断深入,人们逐渐认识到成年CNS再生是由神经元的固有特性(intrinsic properties)和其所处的环境(environmental cues)共同决定的。因此增加受损神经元的再生潜能并改善轴突再生的微环境便成为CNS损伤修复研究的新的楔入点。
神经损伤后修复的难点之一是神经元再生困难,随着近年来干细胞研究的迅猛发展,组织和细胞移植治疗成为学者们最为关注的方法之一,而选择合适的种子细胞则是细胞移植治疗的核心问题。目前用于移植的种子细胞供体主要有胚胎干细胞、神经干细胞(neural stem cells,NSC),以及其他具有成神经分化潜能的成体干细胞。尽管这些细胞在CNS损伤修复移植治疗中均显示出不同程度的积极作用,但由于存在细胞来源局限、取材不易、免疫排斥以及伦理道德等诸多方面的问题,临床应用受到很大限制。因此,寻找新的种子细胞来源成为当务之急。皮肤是人体最大的器官,包括来源于三个胚层的多种不同的组织细胞,自我更新和再生修复速度快,是成体干细胞分离和移植研究的良好对象。真皮多能干细胞(skin-derived precursors,SKPs)是一种新近分离成功的具有高度增殖能力和多向分化潜能的间充质干细胞,具有分化为脂肪细胞、肌细胞及神经细胞的潜能。相对于其他移植供体细胞而言,SKPs具有来源丰富、容易获得、可自体移植和具有更少的“谱系倾向性”等优点,又避免了胚胎伦理学、来源不足和异体排斥等弊端。相关研究表明,SKPs移植治疗脊髓损伤时,不仅能外在补充缺失神经元,同时可通过分化为Schwann细胞促进轴突再生及髓鞘化,显示了SKPs在CNS损伤修复中的良好应用前景。
研究内容和方法:
为了解SKPs移植对脑缺血大鼠中枢神经损伤的修复功能及神经保护作用,本实验进行了下列相关研究。
1.利用悬浮成球及单层贴壁相结合的细胞培养方法,分离成人包皮组织,获得真皮来源细胞,无血清条件下添加B27及生长因子bFGF和EGF使其悬浮生长,培养数日后去除B27及生长因子,添加血清使细胞贴壁生长并快速扩增。获得SKPs细胞后,应用免疫化学方法检测其表面标志物CD29、CD34和Nestin的表达。
2.线栓法制作大鼠大脑中动脉缺血再灌注损伤模型,CM-DiI标记SKPs后,通过股静脉移植到大鼠体内。通过抓握实验、平衡木行走实验及水迷宫实验对实验大鼠进行移植前后神经功能评分。
3.免疫荧光法检测各组实验大鼠神经细胞(NSE、GFAP)相关抗原的表达,观察移植SKPs在实验大鼠体内存活及向神经细胞分化情况。
4.利用TUNEL法分别检测各个时间点缺血对照组及移植组脑内神经细胞凋亡情况,镜下观察脑组织健患侧神经细胞凋亡数目。
结果:
1.采用悬浮成球方法分离培养人真皮来源SKPs,此时细胞扩增较慢,细胞贴壁生长后,细胞扩增速度加快,2-3d传代一次。根据SKPs特异性标志,对其进行免疫染色鉴定,体外单层贴壁培养的真皮多能干细胞,CD29和Nestin呈阳性表达,CD34阴性表达。
2.通过抓握实验、平衡木行走实验及水迷宫实验在SKPs移植后1、3、5、7、14天五个时间点对正常组、脑缺血对照组及SKPs移植组大鼠进行神经功能评分。与正常组比较,缺血后1-14天,模型组各项神经功能检测指标均明显降低(P<0.05);观察期内有自发恢复特点,但仍低于正常组水平:与对照组比较,缺血后3、5、7、14d,SKPs移植组能够增加大鼠的抓握力(P<0.05);促进脑缺血大鼠平衡木行走的能力(P<0.05);与对照组比较缺血后5、7、14d能够明显促进脑缺血大鼠记忆功能的恢复(P<0.05)。
3. SKPs移植1天可见移植的SKPs主要分布在脑组织缺血侧,表明SKPs经股静脉移植后可在短时间内迁移到脑缺血区域,由于我们在免疫组化中应用的是小鼠抗人的GFAP单克隆抗体和兔抗人NSE单克隆抗体,这些表达GFAP和NSE的细胞同时表达CM-DiⅠ,因此GFAP和NSE阳性细胞来源于移植的SKPs,表明SKPs经移植进入实验大鼠体内仍保留向神经细胞分化的特性。为进一步增加脑组织中外源性神经干细胞数量提供了一条思路。
4.正常组和缺血对照组、SKPs移植组的健侧脑组织TUNEL染色只有零星的凋亡细胞。缺血对照组整个缺血区内均可见到凋亡细胞,大量成簇出现的凋亡细胞主要分布在梗死灶边缘区(皮层)的内侧。SKPs移植组凋亡细胞分布与缺血对照组相同,但皮层缺血半暗带区凋亡细胞数(57±15)与缺血对照组(102±7)比较明显减少(P<0.05)。
结论:
我们利用悬浮成球及单层贴壁相结合的培养方法成功地从人包皮组织中分离并扩增了SKPs。移植SKPs到脑缺血损伤大鼠体内能够存活并定向迁移至脑缺血部位并向神经细胞分化,同时可减少缺血区的神经细胞凋亡,促进大鼠脑缺血损伤后的神经功能恢复。结果提示移植的SKPs可能是通过向神经细胞的分化、抑制缺血区神经细胞的凋亡来促进缺血损伤后的神经再生和功能重建,SKPs有望成为干细胞移植治疗的新的种子细胞来源。
The recovery of central nervous system(CNS)after injury is all along a difficult problem that puzzles the neuroscientists.In the past,it was widely accepted that once CNS was injured,nothing could be done.Since recent two decades,numerous studies have been carried out by neuroscientists and it is concluded that CNS after injury can be recovered.The course of recovery based on two factors:intrinsic properties and environmental cues.So,it is aimed to promote the regeneration of neurons and provide a possible agreeable microenvironment for regeneration and recovery of CNS injury.
In regenerative medicine,some encourage results have been obtained.It is well known that cell replacement therapies show particular promise in the nervous system,and transplanted embryonic stem cells(ESCs)or neural stem cells (NSCs) have been shown to promote functional recovery in animal models,for example,spinal cord injury (SCI).Many stem cells (such as ESCs and NSCs) can be used to treat a lot of neurological diseases. Although the therapeutic potential of such transplants is clear,a number of problems, such as insufficient cell number, ethical issues, immunosup- pression, and so on,limit their application in clinic.In this background,a new type of stem cells called skin-derived precursors (SKPs) was discovered in adult mammalian skin.They can differentiate into cells of both neuroectodermal and mesodermal lineage,including(perhaps not limited to) neurons,glia,osteogenic cells and adipocytes.Compared with the above cells, SKPs, which own multi-oriented differentiative potentialand self-renewal capability,have an accessible,potentially autologous tissue source and can expand in vitro cell culture.So they have important therapeutic implications.
In order to determin if the SKPs have the effects of promoting the regeneration and function recovery of Ischemic brain injury. a series of studies were carried out as follows: (1) Combination of suspension and adherent culture method , digested adult dermal tissue, Serum-free conditions, Add B27 and growth factors bFGF and EGF to suspended growth. Immunochemistry to detect the surface markers CD29, CD34 and Nestin. (2) Middle cerebral artery occlusion (MCAO) models were produced by obstructing MCA using a single strand nylon thread, CM-DiI labeled cells, Through the femoral vein transplanted into rats. Neurological function was evaluated with beam-walking,bar-grasping and water maze. (3) Immunofluorescence assay (NSE, GFAP)-associated antigen expression of rat nerve cells. To investigate the survival and differentiation of skin-derived precursors transplanted into rat brain.(4) TUNEL kits were used to detect the control group and transplant brain tissue neural cell apoptosis. Microscopic observation of brain tissue in the number of neuronal apoptosis.
Main results are as following:
1. Combination of suspension and adherent culture , According to SKPs specific signs, Most cells expressed CD29 and Nestin instead of CD34b y immunofluorescent staining.
2. ischemia group rats and SKPs transplantation group rats showed severe neurological disfuction compared with sham-operated group(p<0.05). Rats receiving SKPs had a more rapid recovery of beam-walking,bar-grasping and water maze performance than ischemia group rats(p<0.05).
3. Twenty-four hours later, the transplanted SKPs can be detected mainly in the ischemic zone. These suggested that the transplanted SKPs can migrate to the ischemia zone through the femoral vein graft in a short time. The immunofluorescent staining results confirmed a part of NSE and GFAP positive cells expressed CM-DiI also. These data suggested that the number of nerve cells differentiated from SKPs. In order to further increase the number of exogenous neural stem cells provides a train of thought.
4. TUNEL: After 2 hours of MCA occlusion and 24 hours reperfusion, apoptotic cells exhibiting DNA fragmentation increased in the regions of caudoputaminal and cortex, groups of apoptotic cells were primarily localized to the inner boundary zone of the infarct. The number of apoptotic cells of SKPs transplantation group(57±15)decreased significantly compared with ischemia group(102±7)in the penumbra of cortex with light microscopic analysis (x400 field),p<0.05.
Conclusion: Combination of suspension and adherent culture , Skin-derived precursors (SKPs) were isolated from the adult dermal tissue,cultured and identified by their multilineage differentiation capacity. SKPs can survive in the brain of MCAO rats and were mainly distributed in the region around the ischemic focus and remained the differentiation characters of neuron cells. The results demonstrate that SKPs can improve the neurological function follwing focal cerebral ischemia in rats. Manifested as behavioral score was significantly higher、to reduce the number of neuronal apoptosis in Damage zone. The results suggest that transplant SKPs may be through to Differentiat into neural cells and inhibit neuronal apoptosis in ischemic area to promote nerve regeneration and functional reconstruction after ischemic injury,For clinical treatment of ischemic brain injury provides a new way.
引文
[1] Ramalho-Santos M, Yoon S, Matsuzaki Y, et al.“Stemness”: transcriptional profiling of embryonic and adult stem cells. Science, 2002; 298(5593):597 ~600.
[2] Karl J.L. Fernandes and Freda D. Miller Isolation, Expansion, and Differen- tiation of Mouse Skin-Derived Precursors. Stem Cell , 2009 ; 482 :159~170.
[3] Karl J. L. Fernandes, Jean G. Toma and Freda D. Miller Multipotent skin-der ived precursors: adult neural crest-related precursors with herapeutic potential Phil. Trans. R. Soc. B (2008) 363, 185–198.
[4] Joannides A, Gaughwin P, Schwiening C et al. Efficient generation of neural precursors from adult human skin: astrocytes promote neurogenesis from skin-derived stem cells. Lancet, 2004; 364: 172~178.
[5] Jeff Biernaskie, Joseph S. Sparling, Jie Liu Skin-Derived Precursors Generate Myelinating Schwann Cells That Promote Remyelination and Functional Recovery after Contusion Spinal Cord Injury The Journal of Neuroscience, September 5, 2007 ? 27(36):9545–9559.
[6]王涛,李露斯,刘雁平真皮多能干细胞移植治疗大鼠脊髓损伤的实验研究西南国防医药2007 ,1(17);8~10
[7] Toma JG, Mahnaz A, Karl J et al. Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nature cell biology, 2001; 3: 778~784.
[8]崔苏萍李悦谢安大鼠真皮多能干细胞分离培养及其多分化潜能特性.中华实验外科杂志2007, 24(8); 1006~1007.
[9] Borlongan C V, Yamamoto M, Takei N, et al. Glial cell survival is enhanced during melatonin-induced neuroprotection against cerebral ischemia [J]. FASEB J, 2000, 14 (10): 1307～1317.
[10] Longa E Z, Weinslein P R, Carlson S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats [J]. Stroke, 1989, 20 (1): 84～91.
[11] Urakawa S, H ida H, Masuda T, et al Environmental enrichment brings a beneficial effect on beam walking and enhances the migration of doublecortin-positive cells following striatal lesions in rats [J] .Neuroscience, 2007, 114: 920-933.
[12] Li Y,Chopp M,Jiang N,et al. Induction of DNA fragmentation after 10 to 120 minutes of focal cerebral ischemia in rats. Stroke.1995,26(7):1252-8.
[13]王维治,罗祖明.神经病学, 2002.第四版: p. 122-127.
[14]周良辅,陈衔城等.现代神经外科学, 2004.第一版: p. 758-765.
[15] WHO, Stroke—1989, Recommendations on Stroke Prevention, Diagnosis, and Therapy. stroke, 1989. 20: p. 1407-1431.
[16]张真,急性缺血性脑卒中临床救治现状及研究进展.中国实用医药, 2007. 2(19): p. 82-83.
[17] Watson BD,Prado R,Dietrich WD,et al.Introduction of reproducible brain infarction by photochamically initiated thrombosis[J].Ann Neurol. 1985, 17: 497-500
[18] Markgraf CG,Kraydieh S,Pardo R,et al.Comparative histopathologic consequences of photothrombotic occlusion of the distal middle cerebral artery in Sprague-Damley and Wistarrats[J].Stroke,1993,24(2):286-293
[19]王伟.严格控制实验条件,建立标准化脑缺血动物模型.中华神经科杂志,1998, 31(5): 261-263
[20]陈春富,郭述苏.鼠局灶性脑缺血模型研究进展.前卫医药杂志,1995,12(2):120-122
[21]雷萍,沈关心,干细胞概述.实用医学进修杂志, 2004. 32: p. 1-5.
[22]林戈,卢光琇,干细胞概述.生命科学, 2006. 18: p. 313-317.
[23] Chen J,Bernreuther C,Dihne M,et al.Cell adhesion molecule 11-transfected embryonic stem cells with enhanced survival support regrowth of corticospinal tract axons in mice after spinal cord injury.J Neurotrauma,2005,22(8):896-906.
[24] Hamada M,Yoshikawa H,Ueda Y,et al.Introduction of the MASH1 gene into mouse em- bryonic stem cells leads to differentiation of motoneuron precursors lacking Nogo rece- ptor expression that can be applicable for transplantation to spinal cord injury.Neurobiol Dis,2006,22(3):509-522.
[25] Mauney JR,Kaplan DL,Volloch V.Matrix-mediated retention of osteogenic differentiati- on potential by human adult bone marrow stromal cells during ex vivo expansion.Bio- materials, 2004,25(16):3233-3243.
[26] Jones DG,Anderson ER,Galvin KA.Spinal cord regeneration:Moving tentatively towards new perspectives.NeuroRehabilitation,2003,18(4):339-351.
[27] Young HE,Steele TA,Bray RA,et al.Human reserve pluripotent mesenchymal stem cells are present in the connective tissues of sketetal,muscle and dermis derived from fetal,adult and geriatric donor.Anat Rec,2001,264:51-62.
[28] Cotter,D.C.,Sekiya,L,and Prockop,D.J.Identification of a subpopulation of rapidly self- renewing and multipotential adult stem cells in colonies of human marrow stromal cells.Proc.Natt.Acad.Sci.USA,2001,98:7841-7845.
[29] Young HE,Steele TA,Bray RA,et al.Human reserve pluripotent mesenchymal stem cells are present in the connective tissues of skeletal muscle and dermis derived from fetal,adult,and geriatric donors.Anat Rec,2001,64(1):51-62.
[30] Neuhuber,B.,Gallo,G.,Howard,L.,et al.Reevaluation of in vitro differentiation protocols for bone marrow stromal cells:disruption of actin cytoskeleton induces rapid morphological changes and mimics neuronal phenotype.J.Neurosci.Res.2004,77, 192–204.
[31] Halata,Z.,Grim,M.&Christ,B.Origin of spinal cord meninges,sheaths of peripheral nerves, and cutaneous receptors including Merkel cells.An experimental and ultrastructural study with avian chimeras.Anat.Embryol.(Berl)1990,182,529–537.
[32] Su BY,Cai WQ,Zhang CG,et al.The expressin of ccn3(nov)RNA and protein in the rat central nervous system is developmentally regulated.Mol Pathol,2001, 54(3):184-191.
[33] Nurse,C.A.,Macintyre,L.&Diamond,J.Reinnervation of the rat touch dome restores the Merkel cell population reduced after denervation.Neuroscience 1984,13,563–571.
[34] Nurse,C.A.,Macintyre,L.&Diamond,J.Reinnervation of the rat touch dome restores the Merkel cell population reduced after denervation.Neuroscience,1984,13, 563–571.
[35]李成仁,李巍,蔡文琴,等.NOV对大鼠神经干细胞增殖和分化的影响[J].第三军医大学学报,2003,5(1):15-18.
[36]刘雁平,李露斯,王涛,等。真皮来源间充质干细胞的分离培养及成神经分化的研究[J].西南国防医药,2008,18(1):1-3.
[37] Reynolds,A.J.&Jahoda,C.A.Cultured dermal papilla cells induce follicle formation and hair growth by transdifferentiation of an adult epidermis.Development,1992,115, 587–593.
[38] Reynolds,B.A.&Weiss,S.Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.Science,1992,255,1707–1710.
[39] Reynolds,B.A.,Tetzlaff,W.&Weiss,S.A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes[J].Neurosci,1992,12: 4565–4574.
[40] Toma JG,McKenzie IA,Bageli D,et a1.Isolation and characterization of multipotent skin.derived precursors from human skin.Stem Cell,2005.23:727-737.
[41]柯昌能,利天增,徐盈斌,等.老年人皮肤多能干细胞的分离及有效扩增.中华实验外科杂志,2005,22:1436-1437.
[42]杨林,朱剑虹,王宇倩.绿色荧光蛋白标记神经干细胞的体外研究[ J ].中华实验外科杂志, 2004, 21 (10) : 85 - 87.
[43] Kobayashi T, Ochi M, Yanada S, et al. A novel cell delivery system using magnetically labeled mesenchymal stem cells and an external magnetic device for clinical cartilage repair [ J ]. Arthroscopy, 2008, 24 (1) : 69 - 76.
[44] Hemmrich K, MeerschM, von HeimburgD, et al. App licability of the dyes CFSE, CM-D iI and PKH26 for tracking of human p readipocytes to evaluate adipose tissue engineeri- ng[ J ]. Cells TissuesOrgans, 2006, 184 (3 - 4) : 117 - 127.
[45] Lin D, Najbauer J, Salvaterra PM, et al. Novel method for visualizing and modeling the spatial distribution of neural stem cellswithin intracra2 nial glioma[ J ]. Neuro Image, 2007, 37 ( Supp l 1) : S18 - S26.
[46] Wang L,Li Y,Chen J,et al.Tschemicc erebralt issue and MCP-1 enhance rat bone marrow stromal cell migration in interface culture.Fxp Hematol,2002,30:831-836.
[47] Wang L,Li Y,Chen X,et al.MCP-1,MIP-1,IL-8 and ischemic cerebral tissue enhance hu- man bone marrow stromal cell migration in interface culture.Hematology.Stroke,2002, 7:113-117.
[48] Brook GA,Plate D,Franzen R,et al. Spontnaeous longitudinally orientatedaxonal regeneration is assoeiated with the Schwann cell framework within the lesion site following spinal cord compression injury of the rat.Neurosci Res,1998,53(1):51一65.
[49] Kalla R,Liu Z,Xu S,et al.Microglia and the early phase of immune survivallance in the axotomizes facial motor nucleus:impaired microglial activation and lymphocyte recruitment but no effect on neuronal survival or axonal regeneration in macrophage-colony stimulatingfactor-daficient mice.J Comp Neurol,2001,436:57-65.
[50] Aldskogius H,Kozlova EN.Central neuron-glia and glial-glial interactions following axon injury.Progress in Neurobiology,1998,55:126-136.
[51]胡俊勇,李佛保,廖威明等.大鼠脊髓损伤后神经丝蛋白及胶质纤维酸性蛋自表达的变化及意义,中山大学学报(医学科学版),2003,24(2):129-132.
[52]张卫红,王道新.脊髓损伤后胶质纤维酸性蛋白的表达及其意义.南京医科大学学报,2002,22(l):17一20.
[53] Kerr JFR,Wyllie AH ,Currie AR.Apoptosis:A Basicbiological Phenomenon with Wideranging Implications in Tissun Kinetics.Br J Cancer.1972,26:239-257
[54] Kirino T .Delayed neuronal death in the gerbil hippocampus following ischemia .Brain Res.1982,239(1) :57-69.
[55] Goto K,Ishige A,Sekinguchi k,et al.Effects of cycloheximide on delayed neuronal death in rat hippocampus.Brain Res.1990,534(1-2):299-302.
[56] Okamoto M,Matsumoto M,Ohtsuki T,et al.Internucleosomal DNA cleavage involved in ischemia-induced neuronal death. Biochem Biophys Res Commun 1993,196(3):1356-62.
[57] Rosenbaum DM,Michaelson M,Batter DK,et al. Evidence for hypoxia-induced, programmed cell death of cultured neurons. Ann Neurol. 1994,36(6):864-70.
[58] Ziv Y,Avidan H,Pluchino S,et al.Synergy between immune cells and adult neural stem/ progenitor cells promotes functional recovery from spinal cord injury.Proc Natl Acad Sci U S A,2006,103(35):13174-13179.
[59] Guo JS,Zeng YS,Li HB,et al.Cotransplant of neural stem cells and NT-3 gene modified Schwann cells promote the recovery of transected spinal cord injury[J].Spinal Cord,2007, 45(1):15-24.
[1] Jean G. Toma, Mahnaz Akhavan. Isolation of multipotent adult stem cells from the dermis of mammalian skin [J]. Nature, 2001,126(3): 779 -784.
[2] Jean G. Toma,a Ian A. McKenzie,.Isolation and Characterization of Multipotent Skin-Derived Precursors from Human Skin [J]. Stem Cell, 2005,23(2):727-737
[3]王涛,李露斯,刘雁平真皮多能干细胞移植治疗大鼠脊髓损伤的实验研究西南国防医药2007,1(17);8~10
[4] Joannides A, Gaughwin P, Schwiening C et al. Efficient generation of neural precursors from adult human skin: astrocytes promote neurogenesis from skin-derived stem cells. Lancet, 2004; 364: 172~178.
[5] Fernandes KJL, Kobagashi NR, Gallagher CJ et al. Analysis of the eurogenic potential of multipotent skin-derived precursors. Exp Neuro, 2006; 201: 32~48
[6] Schenke-Layland K, Rhodes KE, Angelis E, et al.Reprogrammed ouseFibroblasts Differentiate into Cells of the Cardiovascular and Hematopoietic ineages[J].Stem Cells, 2008, 26: 1537-1546.
[7] Mauritz C, Schwanke K,Reppel M, et al. Generation of Functional Murine Cardiac Myocytes From Induced Pluripotent Stem Cells[J]. Circulation, 2008, 118(5): 507-517.
[8] Kim JB, Zaehres H, Wu GM, et al. Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors[J]. Nature, 2008, 454(7204): 646-650.
[9] Aoi T, Yae K, Nakagawa M, et al. Generation of Pluripotent Stem Cells from Adult Mouse Liver and Stomach Cells[J]. Science, 2008, 321(5889): 699-702.
[10] Wernig M, Zhao JP, Pruszak J, et al. Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson's disease[J]. Proc Nat Acad Sci U S A, 2008,105(15): 5856-5861.
[11] Dimos JT, Rodolfa KT, Niakan KK, et al. Induced Pluripotent Stem Cells Generated from Patients with ALS Can Be Differentiated into Motor Neurons[J]. Science, 2008, 321: 1218-1221.
[12] Narazaki G, Uosaki H, Teranishi M, et al. Directed and Systematic Differentiation of Cardiovascular Cells From Mouse InducedPluripotent Stem Cells[J].Circulation, 2008,118: 498-506.
[13] Hanna J, Werning M, Markoulaki S, et al. Treatment of Sickle Cell Anemia Mouse Model with iPS Cells Generated from Autologous Skin[J]. Science, 2007, 318(5858): 1920-1923.
[14] Xie C, Huang H, Wei S, et al. A Comparison of Murine Smooth Muscle Cells Generated from Embryonic versus Induced Pluripotent Stem Cells [J]. Stem Cells Dev, 2008:1-32.
[15] Tateishi K, He J, Taranova O, et al. Generation of Insulin-secreting Islet-like Clusters from Human Skin Fibroblasts[J]. J Biol Chem, 2008, 283(46): 31601-31607.
[16] Park IH, Zhao R, West JA, et al. Reprogramming of human somatic cells to pluripotency with defined factors[J]. Nature, 2008, 451(7175): 141-146.
[17] Alexander Meissner, M.W.R.J., Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells[J]. Nat Biotechnol, 2007, 25(10): 1177-1181.
[18] Liao J, Wu Z,Wang Y, et al. Enhanced efficiency of generating induced pluripot-ent stem (iPS) cells from human somatic cells by a combination of six transcript-ion factors. Cell Res, 2008, 18(5): 600-603.
[2] Karl J.L. Fernandes and Freda D. Miller Isolation, Expansion, and Differen- tiation of Mouse Skin-Derived Precursors. Stem Cell , 2009 ; 482 :159~170.
[3] Karl J. L. Fernandes, Jean G. Toma and Freda D. Miller Multipotent skin-der ived precursors: adult neural crest-related precursors with herapeutic potential Phil. Trans. R. Soc. B (2008) 363, 185–198.
[4] Joannides A, Gaughwin P, Schwiening C et al. Efficient generation of neural precursors from adult human skin: astrocytes promote neurogenesis from skin-derived stem cells. Lancet, 2004; 364: 172~178.
[5] Jeff Biernaskie, Joseph S. Sparling, Jie Liu Skin-Derived Precursors Generate Myelinating Schwann Cells That Promote Remyelination and Functional Recovery after Contusion Spinal Cord Injury The Journal of Neuroscience, September 5, 2007 ? 27(36):9545–9559.
[6]王涛,李露斯,刘雁平真皮多能干细胞移植治疗大鼠脊髓损伤的实验研究西南国防医药2007 ,1(17);8~10
[7] Toma JG, Mahnaz A, Karl J et al. Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nature cell biology, 2001; 3: 778~784.
[8]崔苏萍李悦谢安大鼠真皮多能干细胞分离培养及其多分化潜能特性.中华实验外科杂志2007, 24(8); 1006~1007.
[9] Borlongan C V, Yamamoto M, Takei N, et al. Glial cell survival is enhanced during melatonin-induced neuroprotection against cerebral ischemia [J]. FASEB J, 2000, 14 (10): 1307～1317.
[10] Longa E Z, Weinslein P R, Carlson S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats [J]. Stroke, 1989, 20 (1): 84～91.
[11] Urakawa S, H ida H, Masuda T, et al Environmental enrichment brings a beneficial effect on beam walking and enhances the migration of doublecortin-positive cells following striatal lesions in rats [J] .Neuroscience, 2007, 114: 920-933.
[12] Li Y,Chopp M,Jiang N,et al. Induction of DNA fragmentation after 10 to 120 minutes of focal cerebral ischemia in rats. Stroke.1995,26(7):1252-8.
[13]王维治,罗祖明.神经病学, 2002.第四版: p. 122-127.
[14]周良辅,陈衔城等.现代神经外科学, 2004.第一版: p. 758-765.
[15] WHO, Stroke—1989, Recommendations on Stroke Prevention, Diagnosis, and Therapy. stroke, 1989. 20: p. 1407-1431.
[16]张真,急性缺血性脑卒中临床救治现状及研究进展.中国实用医药, 2007. 2(19): p. 82-83.
[17] Watson BD,Prado R,Dietrich WD,et al.Introduction of reproducible brain infarction by photochamically initiated thrombosis[J].Ann Neurol. 1985, 17: 497-500
[18] Markgraf CG,Kraydieh S,Pardo R,et al.Comparative histopathologic consequences of photothrombotic occlusion of the distal middle cerebral artery in Sprague-Damley and Wistarrats[J].Stroke,1993,24(2):286-293
[19]王伟.严格控制实验条件,建立标准化脑缺血动物模型.中华神经科杂志,1998, 31(5): 261-263
[20]陈春富,郭述苏.鼠局灶性脑缺血模型研究进展.前卫医药杂志,1995,12(2):120-122
[21]雷萍,沈关心,干细胞概述.实用医学进修杂志, 2004. 32: p. 1-5.
[22]林戈,卢光琇,干细胞概述.生命科学, 2006. 18: p. 313-317.
[23] Chen J,Bernreuther C,Dihne M,et al.Cell adhesion molecule 11-transfected embryonic stem cells with enhanced survival support regrowth of corticospinal tract axons in mice after spinal cord injury.J Neurotrauma,2005,22(8):896-906.
[24] Hamada M,Yoshikawa H,Ueda Y,et al.Introduction of the MASH1 gene into mouse em- bryonic stem cells leads to differentiation of motoneuron precursors lacking Nogo rece- ptor expression that can be applicable for transplantation to spinal cord injury.Neurobiol Dis,2006,22(3):509-522.
[25] Mauney JR,Kaplan DL,Volloch V.Matrix-mediated retention of osteogenic differentiati- on potential by human adult bone marrow stromal cells during ex vivo expansion.Bio- materials, 2004,25(16):3233-3243.
[26] Jones DG,Anderson ER,Galvin KA.Spinal cord regeneration:Moving tentatively towards new perspectives.NeuroRehabilitation,2003,18(4):339-351.
[27] Young HE,Steele TA,Bray RA,et al.Human reserve pluripotent mesenchymal stem cells are present in the connective tissues of sketetal,muscle and dermis derived from fetal,adult and geriatric donor.Anat Rec,2001,264:51-62.
[28] Cotter,D.C.,Sekiya,L,and Prockop,D.J.Identification of a subpopulation of rapidly self- renewing and multipotential adult stem cells in colonies of human marrow stromal cells.Proc.Natt.Acad.Sci.USA,2001,98:7841-7845.
[29] Young HE,Steele TA,Bray RA,et al.Human reserve pluripotent mesenchymal stem cells are present in the connective tissues of skeletal muscle and dermis derived from fetal,adult,and geriatric donors.Anat Rec,2001,64(1):51-62.
[30] Neuhuber,B.,Gallo,G.,Howard,L.,et al.Reevaluation of in vitro differentiation protocols for bone marrow stromal cells:disruption of actin cytoskeleton induces rapid morphological changes and mimics neuronal phenotype.J.Neurosci.Res.2004,77, 192–204.
[31] Halata,Z.,Grim,M.&Christ,B.Origin of spinal cord meninges,sheaths of peripheral nerves, and cutaneous receptors including Merkel cells.An experimental and ultrastructural study with avian chimeras.Anat.Embryol.(Berl)1990,182,529–537.
[32] Su BY,Cai WQ,Zhang CG,et al.The expressin of ccn3(nov)RNA and protein in the rat central nervous system is developmentally regulated.Mol Pathol,2001, 54(3):184-191.
[33] Nurse,C.A.,Macintyre,L.&Diamond,J.Reinnervation of the rat touch dome restores the Merkel cell population reduced after denervation.Neuroscience 1984,13,563–571.
[34] Nurse,C.A.,Macintyre,L.&Diamond,J.Reinnervation of the rat touch dome restores the Merkel cell population reduced after denervation.Neuroscience,1984,13, 563–571.
[35]李成仁,李巍,蔡文琴,等.NOV对大鼠神经干细胞增殖和分化的影响[J].第三军医大学学报,2003,5(1):15-18.
[36]刘雁平,李露斯,王涛,等。真皮来源间充质干细胞的分离培养及成神经分化的研究[J].西南国防医药,2008,18(1):1-3.
[37] Reynolds,A.J.&Jahoda,C.A.Cultured dermal papilla cells induce follicle formation and hair growth by transdifferentiation of an adult epidermis.Development,1992,115, 587–593.
[38] Reynolds,B.A.&Weiss,S.Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.Science,1992,255,1707–1710.
[39] Reynolds,B.A.,Tetzlaff,W.&Weiss,S.A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes[J].Neurosci,1992,12: 4565–4574.
[40] Toma JG,McKenzie IA,Bageli D,et a1.Isolation and characterization of multipotent skin.derived precursors from human skin.Stem Cell,2005.23:727-737.
[41]柯昌能,利天增,徐盈斌,等.老年人皮肤多能干细胞的分离及有效扩增.中华实验外科杂志,2005,22:1436-1437.
[42]杨林,朱剑虹,王宇倩.绿色荧光蛋白标记神经干细胞的体外研究[ J ].中华实验外科杂志, 2004, 21 (10) : 85 - 87.
[43] Kobayashi T, Ochi M, Yanada S, et al. A novel cell delivery system using magnetically labeled mesenchymal stem cells and an external magnetic device for clinical cartilage repair [ J ]. Arthroscopy, 2008, 24 (1) : 69 - 76.
[44] Hemmrich K, MeerschM, von HeimburgD, et al. App licability of the dyes CFSE, CM-D iI and PKH26 for tracking of human p readipocytes to evaluate adipose tissue engineeri- ng[ J ]. Cells TissuesOrgans, 2006, 184 (3 - 4) : 117 - 127.
[45] Lin D, Najbauer J, Salvaterra PM, et al. Novel method for visualizing and modeling the spatial distribution of neural stem cellswithin intracra2 nial glioma[ J ]. Neuro Image, 2007, 37 ( Supp l 1) : S18 - S26.
[46] Wang L,Li Y,Chen J,et al.Tschemicc erebralt issue and MCP-1 enhance rat bone marrow stromal cell migration in interface culture.Fxp Hematol,2002,30:831-836.
[47] Wang L,Li Y,Chen X,et al.MCP-1,MIP-1,IL-8 and ischemic cerebral tissue enhance hu- man bone marrow stromal cell migration in interface culture.Hematology.Stroke,2002, 7:113-117.
[48] Brook GA,Plate D,Franzen R,et al. Spontnaeous longitudinally orientatedaxonal regeneration is assoeiated with the Schwann cell framework within the lesion site following spinal cord compression injury of the rat.Neurosci Res,1998,53(1):51一65.
[49] Kalla R,Liu Z,Xu S,et al.Microglia and the early phase of immune survivallance in the axotomizes facial motor nucleus:impaired microglial activation and lymphocyte recruitment but no effect on neuronal survival or axonal regeneration in macrophage-colony stimulatingfactor-daficient mice.J Comp Neurol,2001,436:57-65.
[50] Aldskogius H,Kozlova EN.Central neuron-glia and glial-glial interactions following axon injury.Progress in Neurobiology,1998,55:126-136.
[51]胡俊勇,李佛保,廖威明等.大鼠脊髓损伤后神经丝蛋白及胶质纤维酸性蛋自表达的变化及意义,中山大学学报(医学科学版),2003,24(2):129-132.
[52]张卫红,王道新.脊髓损伤后胶质纤维酸性蛋白的表达及其意义.南京医科大学学报,2002,22(l):17一20.
[53] Kerr JFR,Wyllie AH ,Currie AR.Apoptosis:A Basicbiological Phenomenon with Wideranging Implications in Tissun Kinetics.Br J Cancer.1972,26:239-257
[54] Kirino T .Delayed neuronal death in the gerbil hippocampus following ischemia .Brain Res.1982,239(1) :57-69.
[55] Goto K,Ishige A,Sekinguchi k,et al.Effects of cycloheximide on delayed neuronal death in rat hippocampus.Brain Res.1990,534(1-2):299-302.
[56] Okamoto M,Matsumoto M,Ohtsuki T,et al.Internucleosomal DNA cleavage involved in ischemia-induced neuronal death. Biochem Biophys Res Commun 1993,196(3):1356-62.
[57] Rosenbaum DM,Michaelson M,Batter DK,et al. Evidence for hypoxia-induced, programmed cell death of cultured neurons. Ann Neurol. 1994,36(6):864-70.
[58] Ziv Y,Avidan H,Pluchino S,et al.Synergy between immune cells and adult neural stem/ progenitor cells promotes functional recovery from spinal cord injury.Proc Natl Acad Sci U S A,2006,103(35):13174-13179.
[59] Guo JS,Zeng YS,Li HB,et al.Cotransplant of neural stem cells and NT-3 gene modified Schwann cells promote the recovery of transected spinal cord injury[J].Spinal Cord,2007, 45(1):15-24.
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